The overall goal of this experiment is to evaluate the effectiveness of biofilm removal using carbon dioxide aerosols without a nitrogen purge. Carbon dioxide aerosols can be used to remove biofilms utilizing momentum transfer and surfactant action over the aerosols. This is an effective and reliable method to remove biofilms in a short period of time, and is also a biofilm gas-phase cleaning technique.
To begin the experiment, use mechanically cut 1-millimeter thick three zero four stainless steel chips, 10 x 10 square millimeters inside. Next, ultrasonically clean the chips in acetone, methanol, and deionized or DI water sequentially. Rinse the chips with flowing DI water for four seconds, then dry the chips using nitrogen gas flow for four seconds.
Retrieve a Pseudomonas putida KT2440 stock stored at 80 degrees Celsius. Thaw the culture at room temperature for one minute, when the top layer of the frozen stock solution turns to slush. Submerge a loop into the melted layer of the stock solution, and streak the bacteria onto a Luria Broth, or LB plate containing 1.5%agar.
Incubate the plate overnight at 30 degrees Celsius. The next day, add 10 milliliters of LB in a 50 mL conical tube. Use a fresh loop to pick a single colony from the plate and inoculate the LB.Incubate the broth in a shaking incubator.
Pick up each of the prepared chips with tweezers and dip them in 70%ethanol five times for 1-2 seconds each to sterilize the surface of each chip. Next, rinse each chip in autoclaved deionized or DI water. Then, in LB sequentially to remove the remaining ethanol.
Place the chips into six well culture plates with two chips in five millileters of LB broth per well. Then, dilute the bacterial culture until the concentration in the LB broth reaches 8 x 10 to the eighth cells per milliliter. Inoculate each well with 50 microliters of the diluted bacterial culture.
Then, incubate the plates. Dip the biofilmed form chips in 10 millimolar ammonium acetate buffer five times to remove loosely attached and planktonic bacteria. Then, dry the chips in a biosafety cabinet where air flows mildly.
Immediately after drying, place a chip on the loading place, 20 millileters from the carbon dioxide nozzle along the axis of the jet. Tilt the jet axis to a 40 degree angle. Next, set the stagnation pressures of carbon dioxide and nitrogen gas using gas pressure regulators.
Apply the aerosol jet onto the central part of the chip. Turn on the solenoid valve for carbon dioxide for five seconds, and then turn it off for three seconds periodically using a manually controlled switch. If a nitrogen purge needs to be used, turn on the solenoid valve for a continuous supply of nitrogen.
Once the setup is complete, treat the chips with carbon dioxide aerosols with and without nitrogen purges and compare chips with different aerosol treatment times. Retain the chips without treatment as negative controls. Prepare one micromolar of green fluorescence nucleic acid stain in DI water to stain bacterial cells on the control and aerosol treated chips.
Put the staining solution into the wells with the chips, and incubate them. Following the incubation, gently rinse the chips with flowing DI water to remove excessive fluorescent dye. Then, dry the chips with nitrogen gas flow.
Take fluorescence microscopic images of five random fields of view for each chip using an epifluorescence microscope. Finally, move on to calculations. Fluorescence microscopic images for 24 hour grown Pseudomonas putida biofilms were treated with carbon dioxide aerosols for zero, 16, 40, and 88 seconds without nitrogen purges.
Removal efficiencies calculated based on the fluorescence intensities of the biofilms without nitrogen purges were higher than removal efficiencies with nitrogen purges. As you have seen in this video, the tri-phase cleaning technique effectively removes biofilm in a short period of time. It can be applied to a wide variety of bio-contaminated surfaces.
